Sensitivity and Specificity of In situ Proximity Ligation for Protein Interaction Analysis in a Model of Steatohepatitis with Mallory-Denk Bodies

The in situ proximity ligation assay (isPLA) is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs). These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). We investigated the colocalization of all three key MDB components, namely keratin 8 (K8), keratin 18 (K18), and p62 (sequestosome 1) by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8(-/-) and Krt18(-/-) mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol

Search and seizure and its lawfulness in criminal proceedings

Bakalaura darba tēma ir “Kratīšana un tās tiesiskums kriminālprocesā”. Kratīšana ir viena no izmeklēšanas darbībām, kuras saturs ir telpas, apvidus teritorijas, transportlīdzekļa un atsevišķas personas piespiedu pārmeklēšana, nolūkā atrast un izņemt meklējamos objektus, ja ir pietiekams pamats uzskatīt, ka meklējamais objekts atrodas kratīšanas vietā. Ar kratīšanas palīdzību tiek sasniegts taisnīgs kriminālprocesa likuma noregulējums, taču šīs darbības laikā tiek aizskartas cilvēka pamattiesības, līdz ar to darba mērķis ir noskaidrot, kā var veikt kratīšanu nepārkāpjot pamattiesības, un vai tās Latvija tiek ievērotas. Darbā uzmanība pievērsta tam, kā kratīšanas definīcija atšķiras trijās valstīs (Latvijā, Krievijā, Amerikas Savienotas Valstīs (turpmāk – ASV)). Tiks apskatīta katra kratīšanas objekta būtība, ka arī izskatīts kratīšanas pamats jeb kādi lēmumi nepieciešami, lai varētu veikt kratīšanu un vai vispār tie ir vajadzīgi. Tiks noskaidrota kāda ir kratīšanas kārtība un vai tā atšķiras dažādiem kratīšanas objektiem. Kā arī darbā tiks pētīta kratīšanas rezultātu fiksēšana. Darba pamatā tiks izmantots spēka esošais Kriminālprocesa likums un citu valstu normatīvie akti, zinātnieku publikācijas, grāmatas. Darbā analizēta Latvijas un ārvalstu tiesu prakse. Pētījumā tiks izvirzīti secinājumi attiecība uz izskatīto tēmu.Bachelor’s work thesis is – search and seizure and its lawfulness in criminal proceedings. Search and seizure is one of investigative steps whose goal is the compulsory search of a room, teritorry, vehicle or a person with the goal of seizing the sought after object if there is enough evidence to suggest that this object is to be found in the searchable location. Search and seizure helps in achieving just regulation of criminal procedings, but it is imperative that during these actions no human rights must be overstepped. So the goal of this thesis is to establish how to conclude fair and just search and seizure and wheter in Latvia it is done with the human rights in mind. During this thesis apecial attention will be brought to the differences in the definition of search and seizure in three states (Latvia, Russia, USA). Also the meaning of each searchable object will be analyzed. Basis for such a search or – what rulings are needed for the conduction of a search and are they even needed. Also will be determined the procedure of search and whether it even differs with each searchable object and how the results are documented. As the base sources of information will be used Criminal law of Latvia, regulatory enactmens of other states, scientific publications, books, articles and journals. In addition will be used court cases of Latvia and other states. In the thesis there will be highlighted conclusions in relation to the researchable topic

Atypical goblet cell hyperplasia occurs in CPAM 1, 2, and 3, and is a probable precursor lesion for childhood adenocarcinoma

Congenital pulmonary airway malformation (CPAM) is a developmental disorder. Types 1-2-3 are the more common ones. Atypical goblet cell hyperplasia (AGCH) in CPAM might be a precursor lesion for pulmonary adenocarcinomas. In nine out of 33 CPAM cases, types 1-3 showed foci of goblet cell proliferations. As these cells completely replace normal epithelium, we prefer to name these proliferations AGCH. In 5 cases, adenocarcinomas were seen (AC). All cases were analyzed for proteins possibly being associated with CPAM development: fibroblast growth factor 10 (FGF10) and receptor 2 (FGFR2), forkhead box A1 (FOXA1) and A2 (FOXA2), MUC protein 5AC (MUC5AC), human epidermal growth factor receptor 2 (erbB2, HER2/neu), hepatocyte nuclear factor 4α (HNF4α), SOX2, and Ying Yang protein 1 (YY1). By next generation sequencing, AGCH and adenocarcinomas were evaluated for driver mutations. Expression for FGF10, FGFR2, FOXA1, and FOXA2 was seen in CPAM epithelium and stroma, but not differently in AGCH and AC. SOX2 was positive in CPAM epithelium and AGCH, however weakly in AC. YY1 and MUC5AC showed more intense staining in AGCH and AC than in CPAM epithelium. HER2 was intensely expressed in AC and less intensely in AGCH, but not in CPAM epithelium. KRAS mutation in exon 2 was detected in all AGCH and AC, but was absent in CPAM epithelia. AGCH can arise in CPAM types 1-3. Oncogenic KRAS mutation seems to be the oncogenic driver already in AGCH, proving its role as a precursor lesion for adenocarcinoma. It might upregulate HER2 at the protein level. YY1 seems to be involved in carcinogenesis

Testing of ABs to keratin and p62 in mouse liver cells after DDC treatment.

<p>Immunofluorescence stain on SWA mouse livers after 12 weeks DDC treatment using ABs to K8 (green), p62 (red) (<b>A</b>), and K18 (green), p62 (red) ABs (<b>B</b>). For image (<b>A</b>), note the presence of small (arrowhead) and large (empty arrow) MDBs. Additionally, small granules positive for p62 only can be observed (arrow). For image (<b>B</b>), note that small MDB-like keratin aggregates can be observed that are negative for p62 (empty arrow). Scale bars: 20 µm.</p

Schematic drawing of different antibody binding and visualization steps of <i>is</i>PLA combined with IF.

<p>(<b>A</b>) Primary ABs of the <i>is</i>PLA bind to two different proteins, which are expected to colocalize. (<b>B</b>) Secondary ABs conjugated with oligonucleotides bind to the primary ABs (PLA Probe PLUS and PLA Probe MINUS). (<b>C</b>) Two more oligonucleotides (blue) hybridize to the two PLA probes. (<b>D</b>) Ligase (yellow) joins the two added oligonucleotides to form a closed circle. (<b>E</b>) Polymerase (yellow) induces a rolling circle amplification (RCA) using the ligated circle as a template. (<b>F</b>) Fluorescence labeled oligonucleotides (red) hybridize to the RCA product. (<b>G</b>) Primary ABs of the IF (purple) bind to one of the proteins, which is also targeted by <i>is</i>PLA. (<b>H</b>) Secondary ABs of the IF bind to the primary ABs of the IF. They are fluorescence labeled in red while the <i>is</i>PLA signals are green.</p

Influence of channel amplification on demonstration of antigen colocalization in <i>is</i>PLA and IF in 12 week-DDC treated <i>krt18^−/−</i> mice.

<p>(<b>A, B, C</b>) Show different levels of digital amplification of the green channel (IF for K8) and constant amplification of the red channel showing <i>is</i>PLA for K8 and p62. Arrows indicate a MDB-like aggregate that was constantly positive for K8 but negative for p62. Arrowheads indicate MDBs which were positive in <i>is</i>PLA for p62 and K8 but IF showed only presence of K8 (yellow merged signal) in <i>is</i>PLA positive MDBs after maximal amplification of the green channel. Scale bars: 20 µm.</p

<i>is</i>PLA for 12 week-DDC treated mice.

<p>Images (<b>A, B</b>) show <i>is</i>PLA signal (red) using ABs to p62 and K8 (<b>A</b>) and p62 and K18 (<b>B</b>). (<b>C</b>) shows a combined visualization of p62 and K8 in <i>is</i>PLA (red) and K8 in IF (green). (<b>D</b>) shows a combined visualization of p62 and K18 in <i>is</i>PLA (red) and K18 IF (green). In images (<b>C</b>) and (<b>D</b>) note that even very small <i>is</i>PLA positive structures are visible (arrowhead), demonstrating the high sensitivity of <i>is</i>PLA. However, there were also larger MDB-like keratin aggregates that are negative for <i>is</i>PLA stain (empty arrow), suggesting the absence of p62. Scale bars: 20 µm.</p

Technical controls performed on mice with and without DDC treatment.

<p>In order to complement the biological controls (knockout mice) we performed a number of technical controls to make sure that no cross reactions between the different reagents might lead to false results.</p

Testing of ABs to keratin and p62 in mouse liver and demonstration of equivalent binding properties of ABs used in IF and <i>is</i>PLA.

<p>IF staining of normal mouse liver tissues (Swiss Albino, SWA fed a standard diet) with antibodies to K8 (green), p62 (red) (<b>A</b>) and for K18 (green) and p62 (red) (<b>B</b>). Image (<b>C</b>) shows that the two different ABs directed to K8 (rat-anti-K8 [Troma I] and mouse-anti-K8 [Ks 8.7 Progen]) used for <i>is</i>PLA and IF or DIIF recognize identical structures in liver from a DDC fed SWA mouse. Scale bars: 20 µm.</p